Alkaline alpha-galactosidase

ABSTRACT

An enzyme isolated from an organism that metabolizes alpha-galactosyl containing saccharides, comprising an alpha-galactosidase (E.C. 3.2.1.22, alpha-D-galactoside galatohydrolase) with optimal activity in the neutral to alkaline pH range, and which hydrolyzes a variety of alpha-galactose containing saccharides, in particular raffinose. The enzyme is preferably a protein monomer and an ex-alpha-galactosidase.

FIELD OF THE INVENTION

The present invention relates generally to enzymes for hydrolysis ofsugars and particularly to an alkaline alpha-galactosidase whichhydrolyzes a broad spectrum of galactosyl-saccharides such as melibiose,raffinose and stachyose and guar gum, at neutral to alkaline pHconditions.

BACKGROUND OF THE INVENTION

The enzyme alpha-galactosidase (E.C. 3.2.1.22; alpha-D-galactosidegalactohydrolase) catalyzes the hydrolysis of the terminal linkedalpha-galactose moiety from galactose-containing oligosaccharides. Theseinclude, for example, the naturally occurring disaccharide melibiose(6-O-alpha-D-galactopyranosyl-D-glucose), the trisaccharide raffinose(O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside)and the tetrasaccharide stachyose(O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-galactopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-2)-beta-D-fructofuranoside).Alpha-galactosidases have potential use in various applications, andsome examples are described by Margolles-Clark et al. (“Threealpha-galactosidase genes of Trichoderma reesi cloned by expression inyeast”, Eur. J. Biochemistry, 240:104-111, 1996). They may hydrolyzealpha-galactose residues from polymeric galactomannans, such as in guargum; modification of guar gum galactomannan with alpha-galactosidase hasbeen used to improve the gelling properties of the polysaccharide(Bulpin, P. V., et al., “Development of a biotechnological process forthe modification of galactomannan polymers with plantalpha-galactosidase”, Carbohydrate Polymers 12:155-168, 1990).Alpha-galactosidase can hydrolyze raffinose from beet sugar syrup, whichcan be used to facilitate the sugar crystallization from molasses, sincethe raffinose presents an obstacle to the normal crystallization of beetsugar (Suzuki et al., “Studies on the decomposition of raffinose byalpha-galactosidase of mold” Agr. Biol. Chem., 33:501-513, 1969).Additionally, alpha-galactosidase can be used to hydrolyze stachyose andraffinose in soybean milk, thereby reducing or eliminating theundesirable digestive side effects which are associated with soybeanmilk (Thananunkal et al., “Degradation of raffinose and stachyose insoybean milk by alpha-galactosidase from Mortierella vinacea” Jour. FoodScience, 41:173-175, 1976). The enzyme can also remove the terminalalpha-galactose residue from other glycans, such as the erythrocytesurface antigen conferring blood group B specificity. This has potentialmedical use in transfusion therapy by converting blood group type B touniversal donor type O (Harpaz et al. “Studies on B-anticenic sites ofhuman erythrocytes by use of coffee bean alpha-galactosidase”, Archivesof Biochemistry and Biophysics, 170:676-683, 1975, and by Zhu et al.“Characterization of recombinant alpha-galactosidase for use inseroconversion from blood group B to O of human erythrocytes”, Archivesof Biochemistry and Biophysics, 327:324-329, 1996).

Plant alpha-galactosidases from numerous sources have been studied andmultiple forms of the enzyme have been described, such as in Keller F.and Pharr D. M., “Metabolism of Carbohydrates in Sinks and Sources:Galactosyl-Sucrose Oligosaccharides”, In: Zamski, E. and Schaffer, A. A.(eds.) Photoassimilate Partitioning in Plants and Crops: Source-SinkRelationships, ch. 7, pp. 168-171, 1996, Marcel Dekker, Publ., N.Y.These can be classified into two broad groups, acid or alkaline,according to the pH at which they show optimal activity. Practically allstudies of alpha-galactosidases have dealt with the acidic forms of theenzyme and these play important roles in seed development andgermination. Alpha-galactosidases with optimal activity at alkaline pHare uncommon in eucaryotic organisms.

Alpha-galactosidases which show preferred activity to the disaccharidemelibiose are often referred to as melibiases. These may have optimalactivity at alkaline pH but are relatively specific to melibiose, withonly little activity and low affinity to the trisaccharide raffinose. Inaddition, they characteristically function as a multimeric protein. Forexample, the bacterial alpha-galactosidase that has been described fromBacillus stearothermophilus (Talbot, G. and Sygusch, J., “Purificationand characterization of thermostable b-mannanase and alpha-galactosidasefrom Bacillus stearothermophilus”, Applied and EnvironmentalMicrobiology, 56:3503-3510, 1990) has over a 15-fold higher activitywith melibiose, as compared to raffinose and functions as a trimer. Thealpha-galactosidase described from Escherichia coli K12 similarly hasonly about 4% of the activity with raffinose as compared to melibiose,with Km values of 60 mM and 3.2 mM, respectively, in addition tofunctioning as a tetrameric protein (Schmid and Schmitt, “Raffinosemetabolism in Escherichia coli K12: purification and properties of a newalpha-galactosidase specified by a transmissible plasmid”, Eur. J.Biochemistry, 67:95-104, 1976). Similarly, the enzyme from Pseudomonasfluorescens H-601 (Hashimoto, H. et al., “Purification and someproperties of alpha-galactosidase from Pseudomonas fluorescens H-601”,Agric. Biol. Chem., 55:2831-2838, 1991) has relative Km values forraffinose and melibiose of 17 and 3.2 mM, respectively, and functions asa tetramer.

There are obvious advantages to the use of a monomer protein with thedesired enzyme activity, as compared to multimeric proteins. This hasclearly been shown, for example, with the alpha-galactosidases from mungbean seeds (del Campillo, E., et al., “Molecular properties of theenzymic phytohemagglutinin of mung bean”, J. Biol. Chem. 256:7177-7180,1981) in which the retrameric form of the enzyme disassociated into themonomeric form during storage, and this was accompanied by loss ofactivity.

The galactosyl-sucrose sugars, stachyose and raffinose, together withsucrose, are the primary translocated sugars in the phloem of cucurbits,which includes muskmelons, pumpkins and cucumber. The very lowconcentrations of raffinose and stachyose in fruit tissues of muskmelonsuggest that galactosyl-sucrose unloaded from phloem is rapidlymetabolized, with the initial hydrolysis by alpha-galactosidase, asdescribed in “Cucurbits”, Schaffer, A. A., Madore, M. and Phan, D. M.,In : Zamski, E. and Schaffer, A. A. (eds.) Photoassimilate Partitioningin Plants and Crops: Source-Sink Relationships, ch. 31, pp. 729-758,1996, Marcel Decker Publ., N.Y.

P.-R. Gaudreault and J. A. Webb have described in several publications,(such as “Alkaline alpha-galactosidase in leaves of Cucurbita pepo”,Plant Sci. Lett. 24, 281-288, 1982, “Partial purification and propertiesof an alkaline alpha-galactosidase from mature leaves of Cucurbitapepo”, Plant Physiol., 71, 662-668, 1983, and “Alkalinealpha-galactosidase activity and galactose metabolism in the familyCucurbitaceae”, Plant Science, 45, 71-75, 1986), a novelalpha-galactosidase purified from young leaves of Cucurbita pepo, thathas an optimal activity at alkaline conditions (pH 7.5). In addition tothe alkaline alpha-galactosidase, they also reported three acid forms ofthe enzyme, and distinct substrate preferences were found for the acidand alkaline forms. Raffinose was found to be the preferred substrate ofthe acidic forms. The alkaline form had high affinity (Km=4.5 mM) andhigh activity (1.58 μmol galactose formed per min per mg protein) onlywith stachyose. It had low affinity for (Km=36.4 mM) and low activity(0.14 μmol galactose formed per min. per mg protein) toward thetrisaccharide raffinose and hydrolyzed melibiose very slowly andtherefore affinity and activity on that sugar was not calculated. Thus,this previously reported alkaline alpha-galactosidase can be describedas having activity at alkaline pH but with only a narrow spectrum ofsubstrates.

A further characteristic of the alkaline alpha-galactosidase from youngleaves of Cucurbita pepo is that alpha-D-galactose, the product of theenzymatic reaction, is a strong inhibitor of the enzyme's activity(Gaudreault and Webb, 1983), similar to many of the acidalpha-galactosidases. Geaudreault and Webb calculated that 6.4 mMgalactose reduced the reaction velocity of alkaline alpha-galactosidaseby 50%, in a reaction mixture containing 7.5 mM pNPG at pH 7.5. Such aninhibition by the product of the reaction (termed “product inhibition”),generally has important physiological significance in metabolism.

Gaudreault and Webb (among others) have suggested that the alkalinealpha-galactosidase, as the initial enzyme in the metabolic pathway ofstachyose and raffinose catabolism, was important in phloem unloadingand catabolism of transported stachyose in the young cucurbit leaftissue. It is likely that alpha-galactosidase similarly plays animportant role in the carbohydrate partitioning in melon plants, and mayhave possible functions for phloem unloading in fruits of muskmelon.Recently, alpha-galactosidase activity at alkaline pH has been observedin other cucurbit tissue, such as cucumber fruit pedicels, young squashfruit and young melon fruit. Results obtained by Pharr and Hubbard(“Melons: Biochemical and Physiological Control of Sugar Accumulation,In: Encyclopedia of Agricultural Science, vol. 3, pp. 25-37, Arntzen, C.J., et al., eds. Academic Press, N.Y., 1994) led them to suggest thatstachyose degradation by alpha-galactosidase took place within pedicelsof fruit of Cucumis sativus, especially in the regions where the pediceljoins the fruit. Recently, Irving et al. (“Changes in carbohydrates andcarbohydrate metabolizing enzymes during the development, maturation andripening of buttercup squash, Cucurbita maxima D. Delica”, J. Amer. Soc.Hort. Sci., 122: 310-314, 1997) reported the developmental changes inalpha-galactosidase activities, measured at acid and alkaline pH, inbuttercup squash (Cucurbita maxima) fruit. They found that at anthesis,alkaline activity was higher than activity at acid pH and that bothactivities declined during fruit development. Chrost and Schmitz(“Changes in soluble sugar and activity of alpha-galactosidase and acidinvertase during muskmelon (Cucumis melo L.) fruit development”. J. ofPlant Physiology, 151:41-50, 1977) reported approximately similaractivities of alpha-galactosidase at acid and alkaline pH in Cucumismelo fruit at the anthesis stage.

However, all of these studies were carried out using the non-specificartificial substrate, p-nitrophenyl alpha-galactopyranoside (pNPG),which indicates alpha-galactosidase activity but does not reflect eitherthe physiological role of the particular enzyme forms, or, moreimportantly, the substrate specificity of the particular enzyme. Thus,the prior art gives no reason to indicate that the above describedalkaline alpha-galactosidase enzyme activity in the fruit pedicel orfruit tissue, which were assayed with pNPG, might in any way be novel.

Furthermore, it is well established that the artificial substrate pNPGoften indicates a higher pH optimum for alpha-galactosidase activitythan that which is observed with the natural substrates. For example,Courtois and Petek (“Alpha-galactosidase from coffee bean”, Methods inEnzymology, vol. 8:565-571, 1966) state that “Withalpha-phenylgalactoside (pNPG) one observes a pH optimum at pH 3.6, anda second more pronounced peak at pH 6.1. Toward other substrates(melibiose, raffinose, planteose and stachyose) the pH curve is flatter,with a maximum between 3.6 and 4.0”. Similar results were observed forthe alpha-galactosidase of Vicia faba seeds (Dey. P. M. and Pridham, J.B., “Purification and properties of alpha-galactosidase from Vicia fabaseeds”, Bioch. J., 113:49-54, 1969).

While it had been thought that alkaline alpha-galactosidase may beconfined to the cucurbit family, which includes the above mentionedsquash, cucumber and melon plants, it has recently been shown byBachmann et al. (“Metabolism of the raffinose family oligosaccharides inleaves of Ajuga reptens L.”, Plant Physiology 105:1335-1345, 1994) thatAjuga reptens plants (common bugle), a stachyose translocator from theunrelated Lamiaceae family also contains an alkalinealpha-galactosidase. This enzyme was partially characterized and foundto have high affinity to stachyose. Also, leaves of the Peperomiacamptotricha L. plant, from the family Piperaceae, showalpha-galactosidase activity at alkaline pH, suggesting that they alsocontain an alkaline alpha-galactosidase enzyme (Madore, M., “Catabolismof raffinose family oligosaccharides by vegetative sink tissues”, In:Carbon Partitioning and Source-Sink Interactions in Plants, Madore, M.and Lucas, W. J. (eds.) pp. 204-214, 1995, American Society of PlantPhysiologists, Maryland). This indicates the possibility that alkalinealpha-galactosidases, including novel enzymes not previously described,may function in other plants that metabolize galactosyl-saccharides, inaddition to the cucurbits.

The use of an acidic form of alpha-galactosidase in biotechnological andindustrial applications presents problems. For example, the use of anacidic form of alpha-galactosidase to remove the galactose-containingoligosaccharides, which include raffinose and stachyose, from soybeanmilk presents a dilemma, as described by Thanaunkul et al.,(“Degradation of raffinose and stachyose in soybean milk byalpha-galactosidase from Mortierella vinacea”Jour. Food Science,41:173-175, 1976). The pH of soybean milk, which is 6.2-6.4, is wellabove the optimum pH range of the Mortariella vinacea enzyme, which is4.0-4.5, as shown using the natural substrate melibiose. Lowering the pHof the soybean milk solution to conform to the acidic enzyme's pHoptimum caused the soybean proteins to precipitate and left a sour tasteto the milk.

The use of alpha-galactosidase with an acidic pH optimum for the removalof raffinose from beet sugar faces a similar problem. In Suzuki et,1969, (“Studies on the decomposition of raffinose by alpha-galactosidaseof mold” Agr. Biol. Chem., 3-501-513, 1969) the pH of the beet molasseshad to be lowered to 5.2 with sulfuric acid in order for the Mortariellavinacea enzyme to function.

Similarly, seroconversion of blood type B to blood type O would benefitfrom an alpha-galactosidase that is active at neutral to alkaline pH.since the described procedure (Goldstein et al., “Group B erythrocytesenzymatically converted to group O survive normally in A, B, and Oindividuals” Science, 215:168-170, 1982) requires the transfer ofcentrifuged erythrocytes to an acidic buffer in order for the enzyme tofunction. Lowering the pH to the optimum for the coffee beanalpha-galactosidase caused the cells to be less stable and lysis tooccur. Thus, the seroconversion is carried out at pH 5.6, “reflecting acompromise between red cell viability and optimal alpha-galactosidaseactivity”, as reported in Zhu et al. (“Characterization of recombinantalpha-galactosidase for use in seroconversion from blood group B to O ofhuman erythrocytes”, Archives of Biochemistry and Biophysics,327:324-329,1996). Since the natural pH of blood is in the neutral toalkaline range (pH 7.3) alpha-galactosidase with activity in this pHrange would have obvious advantages.

An additional limitation on the industrial utility of the currentlyavailable alpha-galactosidases is that their activity is frequentlyinhibited by the product of the reaction, galactose. As an example, thereported alkaline alpha-galactosidase from Cucurbita pepo leaves(Geaudreault, P. R. and Webb, J. A. “Partial purification and propertiesof an alkaline alpha-galactosidase from mature leaves of Cucurbitapepo”, Plant Physiol., 71, 662-668, 1983) is strongly inhibited byalpha-galactose and it was calculated that 6.4 mM galactose reduced thereaction velocity by 50%.

Thus, there is a well-established need for an alpha-galactosidase withhigh activity at neutral to alkaline pH and with activity towards abroad spectrum of natural galactose-containing saccharides,particularly, but not exclusively raffinose.

SUMMARY OF THE INVENTION

The present invention seeks to provide a novel alkalinealpha-galactosidase which hydrolyzes a broad spectrum of galactosecontaining compounds, including, but not limited to, melibiose,raffinose, stachyose and guar gum. A novel form of alpha-galactosidase(E.C. 3.2.1.22, alpha-D-galactoside galacrohydrolase) was isolated fromyoung melon fruit mesocarp tissue, purified to homogeneity, asdetermined by SDS-PAGE gel electrophoresis, and characterized. Theenzyme is characterized by optimal activity at neutral to alkaline pH(7-8), together with a broader substrate specificity, as compared topreviously reported alkaline alpha-galactosidases. At minimum, theenzyme hydrolyzes stachyose, raffinose and melibiose and guar gum. Bycontrast, a previously described alkaline alpha-galactosidase, which isquite specific for the tetrasaccharide stachyose, shows low activitytoward, and low affinity for, the trisaccharide raffinose and nodetectable activity against the disaccharide melibiose. The novelalkaline alpha-galactosidase enzyme of the present invention waspurified using techniques of differential protein precipitation,ion-exchange chromatography, gel electrophoresis under native anddenaturing conditions. Its native molecular weight is estimated as 84kDa and its denatured molecular weight is estimated as 79 kDa. It is nota glycoprotein, as determined by the absence of binding to the lectinConcanavalin A. It shows relatively low affinity to the inhibitorgalactose (Ki=13 mM), together with relative insensitivity to theinhibitor. In particular, the enzyme has a high affinity for, andactivity against the substrate raffinose.

These characteristics, particularly the neutral to alkaline activityoptimum, together with the broad substrate specificity and mostimportantly the high affinity for raffinose, distinguish the enzyme frompreviously reported alpha-galactosidases. These very samecharacteristics, impart to this enzyme potential use in such diverseapplications as the seroconversion of type B blood to type O blood, aswell as a host of applications in the food products industry.

There is thus provided in accordance with a preferred embodiment of thepresent invention an enzyme isolated from an organism that metabolizesalpha-galactosyl containing saccharides, comprising analpha-galactosidase (E.C. 3.2.1.22, alpha-D-galactosidegalactohydrolase) with optimal activity in the neutral to alkaline pHrange, and which hydrolyzes a variety of alpha-galactose containingsaccharides, in particular raffinose. The enzyme is preferably a proteinmonomer and an exo-alpha-galactosidase.

In accordance with a preferred embodiment of the present invention thealkaline alpha-galactosidase is isolated from a plant that metabolizesalpha-galactosyl containing saccharides.

In accordance with a preferred embodiment of the present invention thealkaline alpha-galactosidase is derived from tissue of a member of thecucurbit family.

In accordance with a preferred embodiment of the present invention thealkaline alpha-galactosidase is derived from tissue of a melon plant.

Further in accordance with a preferred embodiment of the presentinvention the alkaline alpha-galactosidase is characterized by optimalactivity in the range of pH 7-8.

Additionally in accordance with a preferred embodiment of the presentinvention the alkaline alpha-galactosidase is characterized by highaffinity for the substrate raffinose and relatively low inhibition bygalactose.

There is also provided in accordance with a preferred embodiment of thepresent invention a method for seroconversion of group B erythrocytes togroup O erythrocytes, including providing an alkalinealpha-galactosidase which is hydrolytically active above about pH 7.0,and treating group B erythrocytes with the alkaline alpha-galactosidaseso as to remove alpha-linked terminal galactose residues from the groupB erythrocytes, thereby seroconverting the group B erythrocytes to groupO erythrocytes.

There is also provided in accordance with a preferred embodiment of thepresent invention a method for reducing raffinose and stachyose levelsin soybean milk.

There is also provided in accordance with a preferred embodiment of thepresent invention a method for reducing raffinose and stachyose levelsin other plant products or tissues which contain these compounds.

There is also provided in accordance with a preferred embodiment of thepresent invention a method for modifying the rheological properties ofgalactose-containing gum products.

There is also provided in accordance with a preferred embodiment of thepresent invention a method for reducing raffinose levels in sugarbeetmolasses thereby facilitating the crystallization of sucrose from saidsugarbeet molasses.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood and appreciated more fully fromthe following detailed description. taken in conjunction with thedrawings in which:

FIG. 1 is a simplified graphical illustration of separation of acid andalkaline alpha-galactosidases from melon fruit on ion-exchangechromatography, in accordance with a preferred embodiment of the presentinvention. The protein fraction of 5-50% (w/v) PEG-6000 was applied to acolumn of DEAE-Sepharose 4B and eluted with the indicated lineargradient of 0 to 0.45 M NaCl.

FIG. 2 is a simplified graphical illustration of separation of alkalinealpha-galactosidases forms I and II from melon fruit on ion-exchangechromatography, in accordance with a preferred embodiment of the presentinvention. The fractions active at pH 7.5, represented in FIG. 1, werepooled and applied to a HPLC Mono-Q column and eluted with the indicatedlinear gradient of 0 to 0.4 M NaCl.

FIG. 3 shows a Coomassie blue-stained SDS-polyacrylamide gelelectrophoresis (PAGE) gel showing the denatured alkalinealpha-galactosidase forms I (left) and II (right). Next to each of thepurified proteins is a lane showing the separation of markers of knownmolecular weight, for comparison.

FIGS. 4A, 4B and 4C are simplified graphical illustrations of thekinetics of acid (FIG. 4A), alkaline I (FIG. 4B) and alkaline II (FIG.4C) alpha-galactosidases with pNPG, stachyose, raffinose melibiose assubstrate. The relative specificity of alkaline Form II for stachyose isevident, as compared to the broader specificity of alkalinealpha-galactosidase Form I.

FIGS. 4D-F are simplified graphical illustrations of the kinetics ofacid (FIG. 4D), alkaline I (FIG. 4E) and alkaline II (FIG. 4F)alpha-galactosidases with pNPG as substrate, in the presence of varyingconcentrations of the inhibitor galactose. The relative sensitivity ofthe acid form and the alkaline Form II for galactose is evident, ascompared to the insensitivity of alkaline alpha-galactosidase Form I.

FIG. 5 is a simplified graphical illustrations indicating the extent ofinhibition of the alpha-galactosidases by alternative substrates. Inparticular, it shows that the addition of excess raffinose (80 mM) tothe assay medium containing 10 mM stachyose causes 35% inhibition ofmaximal alkaline Form II activity. The inhibition of the acid form byexcess stachyose is demonstrated, particularly, that 80 mM stachyoseadded to the assay medium of the acid alpha-galactosidase causes 45%inhibition, as measure by the free galactose product. Of note is thatalkaline Form I activity, as measured by the production of freegalactose, does not decrease in the presence of excess stachyose.

FIGS. 6A and 6B are simplified graphical illustrations of the effect ofpH on activity of purified acid and alkaline alpha-galactosidases Form Iand Form II. FIG. 6A illustrates the activity against the naturalsubstrates raffinose (acid and alkaline Form I) or stachyose (alkalineForm II); FIG. 6B illustrates the activity against the syntheticsubstrate pNPG. The buffers used were citrate-phosphate (pH 4.0-7.0),HEPES-KOH (pH 7.0-8.0). and Tris (pH 8.0-8.5). All data were adjustedrelative to maximum activity for each enzyme.

FIG. 7 is a simplified graphical illustration of the effect of reactiontemperature on activity of partial purified acid and alkalinealpha-galactosidases I, II with pNPG as substrate. All data wereadjusted relative to maximum activity for each enzyme.

FIG. 8 is a simplified graphical illustration of the calibration curvefrom which the native molecular weight of the enzymes were determined(Sephacryl G-200).

FIG. 9 is a simplified graphical illustration of activities ofalpha-galactosidases during fruit development from ovary throughmaturation. Citrate-phosphate buffer pH 5.5 and HEPES buffer pH 7.5 wereused for the assays with stachyose or raffinose as substrate. Theactivity at pH 5.5 with raffinose as substrate represents the acid formwhile the activities at pH 7.5 with raffinose or stachyose as substraterepresent the alkaline alpha-galactosidase forms I and II, respectively.Data from fruit younger that 6 days after anthesis are from wholeovaries while only the mesocarp portion of the ovaries were sampled fromfruit 10 days and older. Each value is the mean of results from fourreplicates each of which consisted of one fruit. Vertical bars representstandard errors.

Table 1 illustrates the purification scheme for acid and alkaline I, IIalpha-galactosidases from melon fruit. All the specific activities wereassayed using raffinose (for acid and alkaline Form I) or stachyose (foralkaline Form II) as substrate.

Table 2 illustrates the comparison of the kinetic parameters (K_(m),V_(max) and V_(max)/K_(m)) of acid and alkaline Form I and Form IIalpha-galactosidases from melon fruit, using the natural substratesraffinose, stachyose, and melibiose, and the synthetic substrate pNPG.

Table 3 illustrates the comparison of relatives activities of acid andalkaline Form I and Form II alpha-galactosidases from melon fruit usingthe substrates stachyose, raffinose and melibiose (each 10 mM) and guargum (0.1%, w/v). Activity units for each enzyme was scaled so that the1.00 value represents that enzymes activity on its preferred substrate.

Table 4 lists the partial amino acid sequences of two internal peptides,and the N-terminal peptide from the purified alkalinealpha-galactosidase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is now described in detail.

MATERIALS AND METHODS

Plants of muskmelon (Cucumis melo L.cv C-8 ) were crown under standardconditions in a greenhouse in Beit Dagan, Israel. Female flowers werehand pollinated and tagged at anthesis and fruit load was limited to 1fruit per plant after 10 DAA (days after anthesis). For the study, offruit development, primary fruits were harvested from 3 days prior toanthesis, at anthesis and 1, 2 4, 6, 10, 14, 20, 30 and 45 days afteranthesis (DAA). Whole fruits before 6 DAA, and the inner mesocarp of thefruit tissues after 10 DAA, were thinly sliced and immediately frozen inliquid nitrogen prior to storage at −80 C. Chemicals and enzymes, unlessspecified otherwise, were purchased from Sigma and Boehringer, Mannheim,Germany.

Assays for Alpha-Galactosidase

For routine analysis and monitoring the activity in the purificationsteps, alpha-galactosidase was assayed as described by Smart and Pharr(“Characterization of alpha-galactosidase from cucumber leaves”, PlantPhysiology, 66:731-734, 1980), using p-nitrophenyl alpha-galactoside(pNPG) as substrate. Reaction was initiated by adding 50 ul enzymealiquot to either 200 ul 100 mM pH 5.5 McIlvaine buffer or 100 mM pH 7.5HEPES buffer, containing 5 mM pNPG at 35 C. The reaction was terminatedafter 10 min by adding 1 ml of 5% (w/v) Na₂CO₃ and activity expressed asnmol nitrophenol per min as measured at 410 nm. The hydrolysis of thenatural substrates stachyose, raffinose or melibiose byalpha-galactosidases was measured with 10 mM substrate at pH 5.5 or 7.5as in the assay with pNPG. The assay was started by adding 50 ul enzymepreparation at 35 C and terminated after 10 to 20 min by 2 min boiling.Rates of raffinose and stachyose hydrolysis were estimated bydetermining the amounts of galactose released, as described by Smart andPharr (1980) using an enzyme-coupled reaction with NAD and galactosedehydrogenase (Boehringer Mannheim, E.C. 1.1.1.48).

Purification of Alpha-Galactosidases

In order to separate and characterize the various alpha-galactosidasespresent in melon fruit tissue an initial partial purification wascarried out. Mesocarp tissue (200 g fresh weight) from 10 DAA fruit washomogenized in 200 ml chilled extraction buffer containing 50 mMHEPES-NaOH (pH 7.5), 2 mM MgCl₂, 2 mM EDTA and 5 mM DTT. The homogenatewas filtered through four layers of cheese cloth and centrifuged at18,000 g for 30 min. PEG-6000 was used to precipitate proteins fromcrude extract since there was a significantly irreversible loss of theactivity when (NH₄)₂SO₄ was used. Precipitated proteins were collectedfrom the 5-50% (w/v) PEG-6000 fraction, suspended in 50 ml buffer pH 7.5containing 25 mM HEPES and 1 mM DTT (buffer A) and applied to anion-exchange column (DEAE-Sepharose CL-6B, Pharmacia, 1.2×25 cm)previously equilibrated with buffer A. Unbound protein was eluted withbuffer A and the bound protein was eluted at flow rate of 1 ml/min witha linear gradient of 0 to 0.45 M NaCl in buffer A. Fractions of 3.5 mlwere collected and assayed for alpha-galactosidase activity at pH 5.5 orpH 7.5 with pNPG as substrate.

Purification of the Acid Form of Alpha-Galactosidase

For the partial purification of the acid form of alpha-galactosidase thefractions active at pH 5.5 were pooled and concentrated by reversedialysis against solid sucrose. The concentrated fractions werechromatographed on a gel filtration column (Sephacryl-S 200, Pharmacia,4.5×120 cm), previously equilibrated with buffer A, containing 0.15 MNaCl, at flow rate of 0.5 ml/min. Fractions of 3.5 ml were collected andassayed for alpha-galactosidase activity at pH 5.5, using pNPG assubstrate. The active fractions were pooled and NaCl was added to afinal concentration of 0.5 M prior to loading onto a lectin affinitycolumn, (Concanavalin A-Sepharose 4B, 1×5 cm), previously equilibratedwith buffer A containing 0.5 M NaCl. Unbound proteins were eluted withthe same buffer and bound proteins were eluted with the same buffercontaining 50 mM methyl alpha-D-glucopyranoside. The active fractionswere then desalted by dialysis against buffer A for 12 h with twochanges of the buffer. This enzyme fraction was used for thecharacterization of the acidic form of alpha-galactosidase.

Purification of Alkaline Alpha-Galactosidase

The fractions from the DEAE-Sepharose chromatography which were activeat pH 7.5 were pooled and dialyzed against buffer A for 12 h prior toloading onto an HPLC ion exchange chromatography column (Mono-Q HR 5/5,Pharmacia), previously equilibrated with buffer A. Bound proteins wereeluted with a linear gradient of 0.1 to 0.45 M NaCl and the activefractions were detected using pNPG as substrate at pH 7.5 as describedabove. Two peaks of alkaline alpha-galactosidase activity, labeled I andII, were separated by the Mono-Q chromatography. For the furtherpurification of Form II the active fractions of the peak II werechromatographed on hydrophobic interaction chromatography. The tractionswere pooled, brought to 1 M (NH₄)₂SO₄ and loaded on to a hydrophobicinteraction column (phenyl-Sepharose CL-4B, 0.5×12 cm, Pharmacia)previously equilibrated with buffer A containing 1 M (NH₄)₂SO₄. Theprotein was eluted with a reverse stepwise gradient from 1 to 0 M(NH₄)₂SO₄ with 50 mini intervals in buffer A. Fractions containing theactivity peak were collected and dialyzed for 12 h against buffer A andthe dialysate was concentrated by reverse dialysis against solidsucrose. The enzyme, partially purified by hydrophobic interactionchromatography, was used for the characterization of Form II. The activefractions from the hydrophobic interaction column were further purified.Active fractions were separated electrophoretically using a Mini PrepCell (Bio-Rad Laboratories, Hercules, Calif.) for discontinuousnative-PAGE with 7% polyacrylamide, according to manufacturer'sinstructions. Fractions (0.25 ml/fraction) were assayed at pH 7.5 withpNPG as substrate for the activity. The active fractions were pooled,concentrated by Vivaspin Concentrator (Vivascience LTD, Lincoln,England), and run in a 8% SDS-PAGE. Proteins in the SDS-PAGE wereidentified using Coomassie Blue staining.

The hydrophobic interaction chromatography was not applied to thefractions of peak I as there was a great loss of the activity in(NH₄)₂SO₄ solution. Therefore, the active fractions from the Mono-Qcolumn were used for the characterization of Form I. In addition, theForm I enzyme was further purified. as described in the followingsection.

Further purification of alkaline alpha-galactosidase Form I was carriedout. The fractions of peak I, obtained after mono-Q chromatography, werechromatographed on a hydroxyapatite column (BioGel HTP, Bio-Rad, 0.5×12cm) previously equilibrated with 10 mM Na-phosphate buffer pH 7.0containing 0.5 mM DTT. The enzyme was eluted with a 60 ml linear 10 to100 mM Na-phosphate gradient. The active fractions were pooled andconcentrated by Vivaspin Concentrator. The concentrated protein wasseparated electrophoretically on a non-denaturing PAGE using theMini-Protean II apparatus (Bio-Rad) using 1 mm thick slab gelscontaining 10% acrylamide, according to the procedure of Laemmli (1970).The active band was identified as a yellowish band in activity stainwith 50 mM HEPES pH 7.5 containing 2 mM pNPG at 35 C. Following thenative electrophoresis, the active band was excised, and the protein waseluted with ddH₂O overnight and subjected to electrophoreses in 8%SDS-PAGE.

Amino Acid Sequencing

The Coomassie-stained band of purified alkaline alpha-galactosidase FormI was excised from the 8% SDS-PAGE gel and submitted for amino acidsequencing at the Protein Center of the Technion University, Haifa,Israel. The sequencing operation is as follows. Following geldestaining, the protein band was cut with a razor blade and the proteinin it was reduced with DTT (5 mM) and carboxymethylated “in gel” using10 mM iodoacetamide. The gel was then further destained in 50%acetonitrile with 100 mM ammonium bicarbonate, cut to little pieces anddried in vacuum. The gel pieces were rehydrated with 50 mM phosphatebuffer pH8/100 mM ammonium bicarbonate pH 7.4/0.5 M tris-HCl pH 9.2containing the protease (S. aureus V8 protease, Promega)/Lys-C protease(Boehringer)/modified Trypsin (Promega). After an overnight incubationin 37 C. with shaking, the resulting peptides were eluted from the gelpieces with 60% acetonitrile with 0.1% TFA and analyzed by LC-MS asdescribed below.

The peptides were resolved by reverse phase HPLC on a 1×150 mm VydacC-18 column with a linear gradient of 4-65% acetonitrile in 0.025% TFA,at 1%/min at a flow rate of 40 ul/min. The flow was split post column:about 20% of the sample was microsprayed directly from the HPLC columninto an electrospray iontrap mass spectrometer (LCQ, Finnigan) while 80%was collected manually into microfuge tubes for automated Edmansequencing. The mass spec analysis was done in the positive ion modeusing repetitively a full MS scan followed by MS/MS experiment(collision induced fragmentation) on the most abundant ion selected fromthe mass scan. The MS and MS/MS data from the run was compared to thesimulated proteolysis and fragmentation of the proteins in the “owl”database using the “Sequest” software (J. Eng and J. Yates, Univ. ofWashington). Further identification of the protein was performed bysequencing peptides on the automated Sequencer (Perkin Elmer). Twopeptide fragments, designated as P25 and P35 were sequenced by automatedEdman degradation sequencing.

N-Terminal Peptide Sequencing

After resolving the purified alkaline alpha-galactosidase I on SDS-PAGE,as previously described, the protein was blotted to PVDF membrane(Immobilon-CD, Millipore Co.) using a Bio-Rad blotting apparatus. Thetransfer buffer contained 25 mM Tris, 192 mM Glycine, 20% methanol and0.1 mM sodium thioglycolate. The N-terminal amino acid sequence of thepurified alkaline alpha-galactosidase I was analyzed directly from thePVDF membrane using a Automatic Sequencer (Applied Biosystems),according to manufacturer's instructions.

Enzyme Properties

The optimum pH for each partially purified enzyme was determined usingeither 5 mM pNPG, 10 mM stachyose or 10 mM raffinose as substrates, in100 mM McIlvaine buffer over a pH range of 4 to 7, or 100 mM HEPESbuffer at pH range 7 to 8.5, at 35 C. The substrate specificity of thealpha-galactosidases was tested with pNPG, stachyose, raffinose,melibiose or guar gum (Sigma). Effects of galactose, fructose, glucose,sucrose, malate, citrate and of excessive stachyose, raffinose and pNPGon the enzyme activity were assessed. Km, Vmax values for pNPG,stachyose, raffinose or melibiose were determined by Lineweaver-Burkplots, as were Ki (inhibition) values for D-galactose inhibition.

Determination of the Native Molecular Mass and pI (Isoelectric Point)

The partially purified enzymes were chromatographed on a gel filtrationcolumn (Superdex 200 HR 10/30, Pharmacia Biotech., Uppsala, Sweden),equilibrated with 50 mM Na-phosphate buffer (pH 7.0) containing 0.15 MNaCl and 1 mM DTT. Retention time was compared to that of gel filtrationmarkers (Sigma) for molecular weights 12 kDA to 200 kDA. The estimationof pI was carried out by isoelectric focusing (PHASTGEL IEF, PharmaciaBiotech., Uppsala, Sweden) on high speed gel electrophoresis(PHASTSYSTEMTM, Pharmacia Biotech., Uppsala, Sweden) at pH 4.0-6.5.Proteins were loaded to duplicate gels and focused according to themanufacturer's instructions. One of the gels was stained for proteinusing Coomassie Blue. The duplicate gel was sliced into 1 mm segment andassayed for enzyme activity using pNPG at pH 7.5. Standards with pIvalues of 4.55, 5.2 and 5.85 (Sigma) were used for comparison and the pIof the enzymes were estimated from the calibration curve and thedistance of the active band from the anode.

SDS-PAGE (Polyacrylamide Gel Electrophoresis) and Nondenaturing PAGE

Denaturing SDS-PAGE was carried out using a Bio-Rad Mini-Protean IIapparatus using 1 mm thick slab gels containing 8% acrylamide accordingto the procedure of Laemmli (1970). Gels were stained with Coomassiebrilliant blue R-250 and destained in methanol: acetic acid: watersolution. Molecular mass standards used (Pharmacia) were phosphorylase b(94 kD), albumin (67 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD),trypsin inhibitor (20.1 kD), and alpha-lactalbumin (14.4 kD).Nondenaturing PAGE was run with 1 mm thick slab gels containing 10%acrylamide according to the procedure of Laemmli (1970). The active bandwas identified by incubating the gel in 5 nM pNPG in pH 7.5 HEPES bufferat 35 C. and then visualizing the yellow activity band.

Activities of Alpha-Galactosidases During Fruit Development

The activities of alpha-galactosidases in developing fruits wereestimated in crude extracts with raffinose or stachyose as substrate atpH 5.5 or 7.5. Tissues were homogenized in a chilled mortar with 4volumes of chilled extraction buffer containing 50 mM HEPES-NaOH (pH7.5), 2 mM MgCl₂, 2 mM EDTA and 5 mM DTT. After centrifugation at 18,000g for 30 min the supernatant was desalted with a 5 ml Sephadex G-25column and used as the crude enzyme extract. Enzyme extracts from 10 gof 0 and 10 DAA fruits were also characterized after separation on aMono-Q column. The 3 to 50% PEG-6000 (w/v) fraction from the abovesupernatant was separated on a Mono-Q HR 5/5 column previouslyequilibrated with buffer A, with a linear gradient of 0 to 0.45 M NaCl,as above. Active fractions were detected with the assays using pNPG aswell as stachyose or raffinose as substrates at pH 5.5 and 7.5.

Protein Estimation

Protein was estimated according, to the method of Bradford (1976) usingthe BioRad protein assay and BSA as standard.

RESULTS

Purification of Alpha-Galactosidases

Three forms of alpha-galactosidase were resolved from young melon fruitmesocarp by DEAE-Sepharose ion exchange chromatography, in conjunctionwith Mono-Q chromatography, using pNPG as substrate (FIGS. 1 and 2). Thefirst peak showed higher activity at pH 5.5 than at pH 7.5, while thelatter two peaks both showed activity at pH 7.5 with little activity atpH 5.5. Accordingly, we referred the first peak as an acid form ofalpha-galactosidase and the other two peaks as alkalinealpha-galactosidases Form I and Form II, respectively. The three enzymeforms were partially purified for the purpose of characterization (Table1). Mono-Q ion exchange successfully resolved the two alkaline forms,and hydrophobic interaction chromatography was useful in thepurification of alkaline Form II. After further purification, asdescribed in Table 1, the two alkaline forms were elctrophoresed on adenaturing SDS-PAGE gel and a drawing of a photograph of two purifiedproteins is shown in FIG. 3. The acid alpha-galactosidase bound toConcanavalin A-Sepharose, indicating that it is a glycoprotein, and thiswas a useful step in its purification. Neither alkalinealpha-galactosidase forms I or II bound to Concanavalin A, suggestingthat neither are glycoproteins. The purified enzymes were stable for atleast 2 months when stored at −80° C.

Properties of Alpha-Galactosidases

The three enzymes are distinct with respect to their substratespecificity. The hydrolysis of the natural substrates, melibiose,raffinose and stachyose, were of particular interest to us. All threeenzymes showed Michaelis-Menten kinetics at concentrations up to 40 mMmelibiose, raffinose or stachyose (FIGS. 4A, 4B, 4C). Alkaline Form Iexhibited nearly 2-fold higher activity, as well as higher affinity, toraffinose as compared to either melibiose stachyose. Nevertheless, therewas significant activity towards melibiose and stachyose. In contrast,alkaline Form II was relatively specific to stachyose, with littleactivity toward raffinose or melibiose. The acid alpha-galactosidaseexhibited a preferred specificity and higher activity with raffinose ascompared to stachyose or melibiose.

The affinity constants (Km) and calculated maximal velocities (Vmax) forthe substrates raffinose and stachyose, for each of the threealpha-galactosidases are summarized in Table 2. It can clearly be seenthat the Form I alkaline enzyme is novel with respect to its relativelyhigh affinity to both raffinose and stachyose, in distinction from theForm II alkaline enzyme, which is relatively specific to stachyose. Therelative affinity constants (Km) of the two alkaline forms for thesubstrate raffinose is 1.5 and 26.3 for Forms I and II respectively.

The hydrolysis of guar gum, a complex polysaccharide with terminalalpha-galactose moieties, was also investigated. Guar gum (Sigma, 0.1%w/v) was incubated as substrate with the three enzyme fractions and therelative activity of galactose release was measured and is shown inTable 3. It can be seen that of the two alkaline forms only Form I showssignificant activity towards guar gum.

Hydrolysis of the synthetic substrate pNPG did not give any indicationof natural substrate specificity. The acid form showed the highestaffinity for pNPG but highest maximal activity was observed withalkaline Form II, which had the lowest affinity to pNPG (Table 2). Whenusing pNPG as substrate, the acid alpha-galactosidase was inhibitedabove 5 mM pNPG (FIG. 4A). The two alkaline forms followed MichaelisMenten kinetics up to substrate concentrations of 20 mM pNPG (FIGS. 2B,2C).

The inhibition of alpha-galactosidase activity by galactose isrepresented in FIGS. 4D-F. It can clearly be seen that the Form Ialkaline enzyme is relatively insensitive to inhibition by galactose, ascompared to the other forms. A galactose concentration of 8 mM, in thepresence of 10 nM pNPG, caused a reduction of 65% and 70% in activityfor the acid and Form II alkaline enzymes, respectively, but inhibitedthe activity of the alkaline Form I enzyme by a relatively insignificant8%. The inhibition by galactose was characterized as “competitive” forall three enzymes, as determined by calculations from Lineweaver-Burkeplots, with the acid form showing the strongest affinity for theinhibitor (Ki=0.06 mM galactose). The alkaline Form II also showed astrong affinity for the inhibitor (Ki=1.3 mM), as compared to the Form Ienzyme which showed only low affinity for the inhibitor (Ki=13 mMgalactose).

There was an inhibitory interaction between the substrates raffinose andstachyose when either the acid form or the alkaline Form II were assayed(FIG. 5). For the alkaline Form II, addition of 80 mM raffinose to theassay medium containing 10 mM stachyose caused 35% inhibition of Form IIactivity, as measured by the release of galactose. For the acid form, 80mM stachyose added to the assay medium of the acid alpha-galactosidasecontaining 10 mM raffinose caused a 45% inhibition in free galactoserelease. However, this inhibitory interaction was negligible for thealkaline Form I and the addition of excess amounts of stachyose did notlead to a decrease in released galactose.

The acid form exhibits a narrow pH range of maximal activity, between 5and 5.5, with only approximately 5% of maximal activity at pH 7, whenmeasured with its preferred substrate, raffinose (FIG. 6A). When usingpNPG as substrate the acid form exhibited activity over a broad pHrange, from 4 to 8, with maximal activity at 5.8 and approximately 35%maximal activity remaining at pH 7 (FIG. 6B). Both alkaline forms hadmaximal activity at pH 7.5 with raffinose and stachyose (for Form I andForm II, respectively), which was similar to that with pNPG assubstrate. Form I retained high activity up to pH 8.3, while theactivity of Form II declined already at lower pH and at pH 8.0 there wasalready little activity (FIGS. 6A and 6B).

TABLE 1 Activity Protein Specific activity Yield PurificationPurification step nmol min⁻¹ mg nmol mg⁻¹ protein min⁻¹ % fold Acid FormCrude extract 13018 1152 11 100 — 5-50% PEG fraction 11411 481 24 88 2DEAE-Sepharose 4610 45 102 35 9 Sephadex-200 2998 13 232 23 21 Con-A1646 2.6 627 13 55 Alkaline form I Crude extract 27650 1152 24 100 —5-50% PEG fraction 20730 481 43 75 2 DEAE-Sepharose 9475 35 273 34 11Mono-Q 7974 15 537 29 22 Bio-gel HTP 5103 3.7 1382 19 57 Alkaline formII Crude extract 18433 1152 16 100 — 5-50% PEG fraction 16370 481 34 892 DEAE-Sepharose 10907 58 189 59 12 Mono-Q 8826 17 521 48 33 PhenylSepharose 6FF 5761 2.9 1973 31 123

Both alkaline forms I and II exhibited the highest activity in thetemperature range of 35° to 40° C. and activity was significantlydecreased above 40° C. (FIG. 7). The acid alpha-galactosidase wasrelatively thermophilic, with maximal activity at 50° C., and retained40% of its activity at 70° C. (FIG. 5).

The pI values of the two alkaline forms were estimated at 5.0 and 4.7for the forms I and II, respectively, by activity staining ofisoelectric focusing electrophoresis gels. The molecular weight of thedenatured alkaline forms were estimated at 79 kDA and 92 kDA for Form Iand II, respectively (FIG. 3). The molecular weight of the nativeproteins were 27, 84 and 102 kD for the acid form and alkaline Form Iand Form II respectively (FIG. 8).

Changes of Acid and Alkaline Alpha-Galactosidases During Melon FruitDevelopment

The substrate preferences (Table 2) and pH profiles (FIG. 6A) from thepurified acid and Form I and Form II alkaline alpha-galactosidasesallowed us to measure and estimate their activities even in crudeextracts of melon fruit, using their natural substrates. Very littleoverlap in activity occurs between pH 5.5 and pH 7.5 (FIG. 4A) and, atpH 7.5, the activities of alkaline alpha-galactosidase I and II in thecrude extracts could be distinguished by their respective activitieswhen using raffinose or stachyose as substrate. The activity withraffinose at pH 7.5 is a good indicator of Form I activity since Form IIis relatively specific for stachyose. There should be an overestimationof Form II activity when using stachyose due the hydrolysis of thissubstrate by Form I which is also present in the crude extract.Nevertheless, distinct developmental patterns of alpha-galactosidaseactivities are apparent when using these two substrates.

TABLE 2 Km Vmax α-galactosidase Substrate (mM) (unit*) Vmax/Km Acid formStachyose 10.5 0.23 0.02 Raffinose 4.2 0.71 0.17 Melibiose 0.7 0.19 0.27pNPG 0.3 1.5 5.oo Alkaline I Stachyose 4 0.24 0.06 Raffinose 1.5 0.560.37 Melibiose 20 0.3 0.02 pNPG 1.2 1.4 1.17 Alkaline II Stachyose 3.62.2 0.61 Raffinose 26.3 0.28 o.o1 Melibiose 18.7 0.23 o.o1 pNPG 3 7.92.63 *unit: μmol mg⁻¹ protein min⁻¹

Stachyose hydrolysis at alkaline pH was highest in the pre-anthesisfruit ovary and progressively declined through development (FIG. 9).Raffinose hydrolysis at alkaline pH (Form I), in comparison, increasedduring the pre-anthesis period and remained high during the initialfruit-setting period, declining only from 10 days after anthesis. Themajor alpha-galactosidase activity in the mature fruit was towardraffinose at alkaline pH. Raffinose and stachyose hydrolysis at acid pHalso declined during fruit development but the relative hydrolysis ofthe two substrates remained the same at each stage measured (FIG. 9).

Partial amino acid sequence data was determined for the Form II enzymeas described earlier. Table 4 lists the amino acid sequence for twointernal peptides and the N-terminal peptide. A comparison of the threepeptide sequences against the SwissProt protein sequence database usingthe BLAST program did not reveal any meaningful homologies.

TABLE 3 Acid Alkaline Alkaline Substrate α-galactosidase α-galactosidaseI α-galactosidase II Activity (unit) Stachyose 0.290 0.511 1.000Raffinose 1.000 1.000 0.074 Melibiose 0.274 0.315 0.090 Guargum 0.3280.181 0.032

In summary, although acid alpha-galactosidases often exist in multipleforms in leaves and seeds, only one form of alkaline alpha-galactosidasehas been reported in plants in the prior art (Gaudreault and Webb 1983,1986). In the present invention, three different alpha-galactosidasesextracted from the fruit tissue of muskmelon are demonstrated, asresolved by ion-exchange chromatography (FIGS. 1 and 2). Two of thepurified alpha-galactosidases exhibit maximum activity atneutral-alkaline conditions (FIG. 6). In addition to the pH optima, thetwo alkaline alpha-galactosidases show similar temperature sensitivity,and are non-glycosylated, in contrast to the acid alpha-galactosidase inmelon fruit.

The purified acid form we studied is similar to the smaller molecularform of acid alpha-galactosidase isolated from cucumber leaves (Smartand Pharr, 1980), with respect to pH optima and Km for raffinose orstachyose. The alkaline alpha-galactosidase Form II which we report hereappears similar to the previously reported alkaline alpha-galactosidasefrom squash leaves (Gaudreault and Webb, 1982, 1983), with respect to pHoptima and affinity to stachyose and raffinose, as well as to inhibitionby the product galactose.

The two alkaline alpha-galactosidases can be distinguished from oneanother by a number of characteristics, such as substrate affinities,pI, molecular weight and different inhibition by D-galactose and aninteractive inhibition between the natural substrates, raffinose andstachyose. The most significant difference between the two alkalineforms is in their substrate preferences when hydrolyzing naturalgalactosyl-saccharides. The Form II enzyme is relatively specific forstachyose while the Form I shows preferred activity against raffinose,with significant activity against other galactose containing saccharidessuch as stachyose, melibiose and guar gum, as well.

It is a particular feature of the present invention that the Form Ialkaline alpha-galactosidase has a high affinity for the substrateraffinose, as expressed in the enzyme's Km_(raffinose) which is <5 mM.

Seroconversion of Group B Erythrocytes to Group O Erythrocytes

It is well established that group B erythrocytes can be enzymaticallyconverted to group O erythrocytes in vitro bv using aalpha-galactosidase enzyme. This is because the group B antigen differsstructurally from the group O antigen only by the addition of oneterminal alpha-linked galactose residue.

Such conversion is discussed in “Characterization of Recombinantalpha-Galactosidase for Use in Seroconversion from Blood Group B to O ofHuman Erythrocytes”, A. Zhu et al., Archives of Biochemistry andBiophysics, 327:324-329, 1996. Acid alpha-galactosidase isolated fromgreen coffee beans has been shown in the prior art to be highly activein removing the terminal alpha-linked galactose residues from the groupB red cell surface. Similarly, the prior art shows that acidalpha-galactosidase enzymes from tomato fruit (Pressey, R., “TomatoAlpha-Galactosidase: Conversion of Human Type B Erythrocytes to Type O”Phytochemistry 23:55-48, 1984) and mung beans (Dey, P. M.,“Characteristic Features of an Alpha-Galactosidase from Mung Beans”,Eur. J. Biochem. 140:385-390) can each seroconvert type B erythrocytesto type O erythrocytes. In Zhu et al., a recombinant acidalpha-galactosidase from green coffee beans was produced in Pichiapastoris, a methylotrophic yeast strain, and recombinantalpha-galactosidase was purified from the P. pastoris culturesupernatant by a simple chromatography procedure. The recombinantalpha-galactosidase was used to seroconvert group B erythrocytes togroup O erythrocytes in vitro.

Seroconversion with the above described acid alpha-galactosidase enzymeshas a severe disadvantage. These described alpha-galactosidase enzymesshow optimum activity in the acidic pH range. For example, the enzymedescribed by Zhu et al. is acidic, displaying maximal activity at pH 6.4toward the substrate pNPG, dropping sharply at pH's higher than 7.0 andhaving a second peak at pH 4.5. The optimal pH drops to between 3.6 and4 if the substrate is melibiose, raffinose or stachyose. The removal ofterminal alpha-linked galactose residues from the group B red cellsurface by coffee bean alpha-galactosidase was observed when the pH wasless than 6.0. However, the physiological pH of blood is about 7.3. Zhuet al. thus had to compromise and use recombinant alpha-galactosidase totreat red blood cells at pH 5.5. It is noted that the recombinantalpha-galactosidase exhibited a high activity at pH lower than 5.5, butthe cells were less stable and began to lyse.

As described herein above, the alkaline alpha-galactosidase of thepresent invention is characterized by optimal activity at neutral toalkaline pH (7-8), in contrast to the prior art acidalpha-galactosidases. The enzyme of the present invention also featuresa broader substrate specificity, as compared to the prior art alkalinealpha-galactosidase. Accordingly, the alkaline alpha-galactosidase ofthe present invention may be used to seroconvert croup B erythrocytes ofhuman blood to group O at the natural pH of human blood, and promises tobe more effective than the enzymes used in the prior art since theenzyme's optimal pH encompasses the natural pH of human blood.

Examples of other uses of the alkaline alpha-galactosidase of thepresent invention are in the food industry. Certain legumes, such assoybean and its milk product, contain stachyose and raffinose which ismetabolized in humans only by the microbial flora in the largeintestine, thereby causing problems of flatulence. The pH of soybeanmilk is approximately 6.4 and cannot be lowered due to proteinprecipitation at a lower pH. However, the stachyose and raffinose may beefficiently hydrolyzed by the alkaline alpha-galactosidase of thepresent invention, thereby reducing significantly or eliminatingaltogether problems associated with digestion of these sugars.

TABLE 4 Peptide Amino acid sequences P-25 EYPIQSPGNVSNL (SEQ. ID No. 1)P-35 DISLTE(R/L)VT (SEQ. ID No. 2) N-terminal TVGAGITISDANLTVLG (SEQ. IDNo. 3)

Another use of the alkaline alpha-galactosidase of the present inventionin the food industry, is in the enzymatic hydrolysis of thetrisaccharide raffinose in sugar beet molasses to galactose and sucrose.The presence of raffinose in sugar beet molasses inhibits thecrystallization of the commercially important sucrose in the molasses.The hydrolysis of the raffinose to galactose and sucrose facilitates thecrystallization of the sucrose.

Another use of the alkaline alpha-galactosidase of the present inventionin the food industry, is in the enzymatic modification of plant gums,such as guar gum. The prior art (Bulpin et al., “Development of abiotechnological process for the modification of galactomannan polymerswith plant alpha-galactosidase” Carbohydrate Polymers 12:155-168, 1990)has shown that the modification of guar gum by alpha-galactosidasemodifies the rheological and stabilization properties of the gum, makingit similar to the more functional and more expensive locust bean gum.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed hereinabove. Rather the scope of the present inventionincludes both combinations and subcombinations of the features describedhereinabove as well as modifications and variations thereof which wouldoccur to a person of skill in the art upon reading the foregoingdescription and which are not in the prior art.

3 1 13 PRT Artificial sequence Peptide derived from proteolyticdegredation for protein identification 1 Glu Tyr Pro Ile Gln Ser Pro GlyAsn Val Ser Asn Leu 1 5 10 2 9 PRT Artificial sequence Peptide derivedfrom proteolytic degredation for protein identification 2 Asp Ile SerLeu Thr Glu Xaa Val Thr 1 5 3 17 PRT Artificial sequence Peptidesequence derived from N′- sequencing of alkaline alpha-galactosidase I 3Thr Val Gly Ala Gly Ile Thr Ile Ser Asp Ala Asn Leu Thr Val Leu 1 5 1015 Gly

What is claimed is:
 1. An isolated enzyme from a plant of the Cucurbitfamily comprising, a) an alpha-galactosidase (E.C. 3.2.1.22,alpha-D-galactoside galactohydrolase) activity; b) a K_(m) for thesubstrates raffinose of less than about 10 mM; c) optimal activity inthe range of pH 7.0 to pH 8.0; d) molecular mass of about 84 kDa, asdetermined by gel electrophoresis; the enzyme being non-glycosylated. 2.An enzyme according to claim 1 wherein said K_(m) for the substrateraffinose is less than about 5 mM.
 3. An enzyme according to claim 1wherein said K_(m) for the substrate raffinose is less than or equal toabout 1.5 mM.
 4. An enzyme according to claim 1 wherein said plant is amelon plant.
 5. An enzyme according to claim 1 isolated from the fruittissue of the plant.
 6. An enzyme according to claim 1 wherein saidenzyme is a protein monomer.
 7. An enzyme according to claim 1 whereinsaid enzyme is an exo-alpha-galactosidase.
 8. A method for removingalpha-galactose from galactosyl-saccharide containing materialcomprising: a) providing an enzyme of claim 1; and, b) contacting saidenzyme with said galactosyl-saccharide containing material so as toremove alpha-galactose from said galactosyl-saccharide containingmaterial.
 9. An isolated plant enzyme having, a) an alpha-galactosidase(E.C. 3.2.1.22, alpha-D-galactoside galactohydrolase) activity; b) aK_(m) for the substrates raffinose of less than about 10 mM; c) optimalactivity in the range of pH 7.0 to pH 8.0; d) molecular mass of about 84kDa, as determined by gel electrophoresis; the isolated plant enzymebeing non-glycosylated and having an N-terminal sequence as set forth inSEQ. ID. NO:
 3. 10. An isolated plant enzyme of claim 9 wherein saidK_(m) for the substrate raffinose is less than about 5 mM.
 11. Anisolated plant enzyme of claim 9 wherein said K_(m) for the substrateraffinose is less than or equal to about 1.5 mM.
 12. An isolated plantenzyme of claim 9 isolated from an alpha-galactosyl saccharidemetabolizing plant.
 13. An isolated plant enzyme of claim 12 whereinsaid plant is a member of the Cucurbit family.
 14. An isolated plantenzyme of claim 13 wherein said plant is a melon plant.
 15. An isolatedplant enzyme of claim 9 wherein said enzyme is isolated from the fruittissue of the plant.
 16. An isolated plant enzyme of claim 9 whereinsaid enzyme is a protein monomer.
 17. An isolated plant enzyme accordingto claim 9 wherein said enzyme is an exo-alpha-galactosidase.
 18. Amethod for removing alpha-galactose from galactosyl-saccharidecontaining material comprising: a) providing an isolated plant enzyme ofclaim 9; and, b) contacting said enzyme with said galactosyl-saccharidecontaining material so as to remove alpha-galactose from saidgalactosyl-saccharide containing material.